The addiction to nicotine results in a significantly shortened life span in millions of people and in billions of health care dollars spent every year in tobacco related illnesses. Although the mechanism of addiction is not understood there is a growing body of evidence pointing to the involvement of high-affinity neuronal nicotinic receptors (nAChR) in addiction. Our project will explore possible mechanisms that underlie nicotine addiction. Our studies will be carried out in adrenal chromaffin cells, which may be a primary peripheral target for nicotine. Chromaffin cells express neuronal nAChR similar to those in neurons; the subsequent catecholamine release triggered by nicotine may be involved in the addiction process. Release of catecholamine may affect blood pressure and heart rate and may be involved in some of the adverse physiological changes seen in smokers. In addition, we will study physiological release mechanisms mediated by ACh (or nicotine). We have helped develop a new model system that will allow us to directly test the efficacy of neuronal nicotinic receptors in initiating neurotransmitter release. Similar studies will be carried out in chromaffin cells. The specific aims of this grant are: Aim 1 (Chromaffin Cells) - Hypothesis: Activation of nicotinic receptors directly triggers or modulates neurotransmitter release. Nicotine levels in smokers are sufficient to produce significant desensitization of receptors, which alters release. Because nAChRs have a high permeability to Ca2+ our goal is to determine the importance of this Ca2+-influx pathway in triggering release using physiologically relevant applications of nicotine. These experiments will be repeated after both short- and long-term (20 mm. to 6 days) exposure to low levels of nicotine (100 -- 500 nM) similar to those observed in smokers. Because elevations of intracellular Ca2+ mobilize vesicles for release, we will try to determine whether low levels of nicotine (100 -- 500 nM) affect vesicular mobilization and the role of desensitization in this process. Aim 2 (mouse pheochromcytoma cells) - Hypothesis: Activation of a variety of different nAChR can directly trigger secretion. My lab (in collaboration with Dr. Art Tischler's lab) recently identified a mouse pheochromocytoma (MPC) cell line that is secretion competent but which expresses no nicotinic receptors and few or no endogenous Ca2+ channels. We will transfect these cells with different types of nicotinic receptors to determine whether they can trigger secretion or mobilize vesicles and the mechanisms involved. Subunits that will be tested include a7 (alone), a3 b2, a3 / b4 and a4/b2. For these experiments we will employ cells that express no endogenous Ca2+ currents (determined by briefly depolarizing every cell). Physiologically, activation of nAChRs depolarizes cells and activates Ca2+ channels. In other experiments we will co-transfect nAChRs and the a1b/a2d/b3a(N-type) Ca2+ channel subunits in order to study the synergy between these Ca2+-inf1ux pathways. This new aim is extremely exciting to us and is only possible because of the discovery of this remarkable cell line.